The current monograph requires evidence of efficacy for fluoride toothpaste, typically through animal studies or established in vitro tests. To date, the Animal Caries Reduction Test using rats has also been applicable; this is a biological test method for assessing the effectiveness of fluoride-containing toothpastes and active ingredients against dental caries. In this test, rats are typically infected with caries-promoting bacteria such as Streptococcus mutans and fed a sugar-rich diet to measure the extent of caries formation under the influence of a test toothpaste. The animals’ teeth are then treated with the test toothpaste using a standardized procedure. After a specified period, the animals are euthanized so that their teeth and jaws can be examined microscopically in the laboratory. The caries lesions on the teeth are counted and evaluated, and compared with those of the control group animals, which are also euthanized.

Since the dental physiology of rodents differs from that of humans, modern regulatory approval processes (such as those of the U.S. Food and Drug Administration) are increasingly focusing on in vitro alternatives such as pH-cycling models or micro-computed tomography analyses.

The pH cycle test model is an animal-free method developed to evaluate the effectiveness of fluoride toothpastes in preventing dental caries without the use of animal testing. The model simulates the natural processes in the mouth, in which tooth enamel is demineralized by acid and remineralized by saliva. There are various versions of the pH cycle test, such as the Featherstone model, which uses precisely defined chemical compositions and fixed time intervals. However, a standardized and validated protocol for these versions has been lacking to date. A manufacturer seeking to obtain approval for the pH cycling test as a testing method for its fluoride toothpaste should therefore now develop a standardized test protocol with the FDA, conduct a multicenter validation study, compare the results with established animal studies, and submit all relevant data and analyses in accordance with FDA guidelines.

The Featherstone pH-cycling model is a laboratory test that simulates the natural process of demineralization (loss of minerals) and remineralization (rebuilding of minerals) in teeth. It serves as an animal-free alternative for measuring the protective effect of fluoride-containing toothpastes or mouthwashes against tooth decay. Artificial caries lesions are induced on the tooth samples. The teeth are then briefly immersed in an acidic solution to leach out minerals. Afterward, they are placed in a mineral-rich buffer solution designed to simulate the remineralizing effect of saliva. The samples are briefly treated daily with the substance under test (e.g., toothpaste solutions). The teeth come mainly from human extractions (wisdom teeth, orthodontic extractions, or from dental practices, provided patients consent to making them available for research) and bovine teeth. The reason given is that human teeth are less readily available due to a decline in caries incidence, and hundreds of identical samples are needed for statistically reliable test results.

However, the results of the pH cycle test must be compared with established animal tests to demonstrate equivalence, even though the dental physiology of rodents differs from that of humans.

Situation in Europe
Since toothpaste and mouthwash are legally classified as cosmetics, animal testing is not permitted for their finished products or purely cosmetic ingredients—except in a legal gray area where a new chemical ingredient in toothpaste is manufactured industrially in massive quantities under the European Chemicals Regulation (REACH): in such cases, the law requires tests for work safety (e.g., inhalation toxicity in the factory). Biological research on living organisms is also possible through animal testing. And for that, they may use rats or mice.

Sources:
https://www.accessdata.fda.gov/scripts/cder/omuf/index.cfm?event=datareqDetail&utm_medium=email&utm_source=govdelivery
Featherstone JD, Stookey GK, Kaminski MA, Faller RV. Recommendation for a non-animal alternative to rat caries testing. Am J Dent. 2011 Oct;24(5):289-94. PMID: 22165456. https://pubmed.ncbi.nlm.nih.gov/22165456/
Stookey GK, Featherstone JD, Rapozo-Hilo M, Schemehorn BR, Williams RA, Baker RA, Barker ML, Kaminski MA, McQueen CM, Amburgey JS, Casey K, Faller RV. The Featherstone laboratory pH cycling model: a prospective, multi-site validation exercise. Am J Dent. Oct 2011;24(5):322-8. PMID: 22165462.